Cultivation and Genetic Manipulation of Free-Living and Pathogenic Leptospires

钩端螺旋体病是一种由钩端螺旋体属成员引起的全球性人畜共患病。尽管钩端螺旋体病广泛存在，有时甚至致命，但它被认为是一种被忽视和研究不足的疾病。钩端螺旋体病的病原体于1916年首次被发现，但缓慢的体外生长速度和有限的基因工具操作这种螺旋体的基因组，阻碍了毒力因子的识别和疫苗的开发。 钩端螺旋体大致可分为两类：自由生活的腐生生物和传染性病原体。最广泛使用和研究的物种是双歧杆菌(一种非致病腐生植物)和问号乳杆菌(一种病原体)。然而，与致病性问号钩端螺旋体相比，非致病性双向钩端螺旋体更容易培养，更容易受到遗传操作的影响。因此，我们将重点放在双挠性乳杆菌上，以掌握操纵该属所需的微生物和遗传技术，目的是将这一专业知识转移到更难对付的致病菌株。靶向基因失活、穿梭载体转化和转座子诱变都已在双歧杆菌中得到成功应用。到目前为止，还没有针对致病物种的穿梭载体系统，也很少有问号钩端螺旋体靶向基因失活的报道。转座子诱变可以应用于问号钩端螺旋体，但它的作用效率很低，不能用于任何广泛的应用，如营养缺陷性筛选或标记突变。缺乏问号钩端螺旋体的穿梭载体阻碍了互补，从而限制了对转座子或靶向缺失突变体的任何表型的解释。由于双挠性乳杆菌比其他物种具有更高的转化频率，我们计划优化这种生物的新技术。 在2014财年，我们已经开始评估可能影响钩端螺旋体转化效率的不同系统。Lamda Red重组酶系统已经成功地应用于其他细菌，以提高定向突变。我们已经开始在双弯乳杆菌中评估这个系统，如果它看起来很有希望，我们将在病原体问号钩端螺旋体中进行测试。此外，我们正在研究CRISPR/Cas系统，该系统存在于问号钩端螺旋体中，但不存在于两栖钩端螺旋体中。该系统靶向并降解外源DNA，我们推测这可能是病原菌相对于腐生植物转化频率较低的原因。具体地说，我们已经证明CRISPR/cas操纵子在体外生长过程中转录，并已将部分操纵子整合到双挠性乳杆菌中，并表明这些基因也在这个异源宿主中转录。目前，我们正在尝试灭活问号钩端螺旋体中的特定cas基因，并将整个操纵子转移到双歧钩端螺旋体中。 我们在2014财年开始绘制体外培养的双歧乳杆菌的蛋白质组学图谱，以从膜和可溶部分中鉴定高表达的蛋白质。我们已经确定了大量表达的蛋白质，这些蛋白质可以用作细胞标记，作为基因表达研究的对照，并对这些基因的子集的转录数据进行了量化。此外，我们还证明了大量的双弯乳杆菌蛋白受到翻译后修饰的影响，包括磷酸化和乙酰化。高表达的蛋白质使我们能够识别可能发挥重要生理作用的靶点，并将其用作各种表达研究的标记蛋白。这项工作是在与NIAID持久性病毒疾病实验室的James Carroll博士和NIAID研究技术处的Lisa Olano博士的内部合作下完成的。 该项目的长期目标是使用改进的工具和技术来了解钩端螺旋体的基本生理学以及问号钩端螺旋体的感染和致病机制。总而言之，这些知识应有助于加快制定预防钩端螺旋体病的措施。

Leptospirosis is a global, zoonotic disease caused by members of the genus Leptospira. Although widespread and sometimes fatal, leptospirosis is considered a neglected and understudied disease. The causative agent of Leptospirosis was first identified in 1916 but the slow in vitro growth rate and limited genetic tools with which to manipulate the genome of this spirochete have hampered the identification of virulence factors and development of a vaccine. Leptospires can be broadly divided into two groups: free-living saprophytes and infectious pathogens. The most widely used and studied species are L. biflexa (a non-pathogenic saprophyte) and L. interrogans (a pathogen). However, the non-pathogenic L. biflexa is more easily cultivated and more amenable to genetic manipulation than the pathogenic L. interrogans. Therefore, we have focused on L. biflexa to master the microbial and genetic techniques needed to manipulate this genus, with the intention to transfer this expertise to the more refractory pathogenic strains. Targeted gene inactivation, shuttle vector transformation, and transposon mutagenesis have all been successfully used in L. biflexa. To date, no shuttle vector system exists for pathogenic species and there are few published reports of targeted gene inactivation in L. interrogans. Transposon mutagenesis can be applied to L. interrogans but it functions at such a low efficiency that it cannot be utilized for any broad applications, such as auxotrophic screens or signature tagged mutagenesis. The lack of a shuttle vector for L. interrogans hinders complementation and thus limits interpretation of any resulting phenotypes of transposon or targeted deletion mutants. Since L. biflexa has a better transformation frequency than other species we plan to optimize new techniques in this organism. In FY2014 we have begun to evaluate different systems that may affect the transformation effiencies of leptospires. The lamda red recombinase system has been used successfully in other bacteria to improve targeted mutagenesis. We have begun to assess this system in L. biflexa, and if it appears promising, we will test it in the pathogen L. interrogans. Also, we are studying the CRISPR/Cas system that is present in L. interrogans but absent in L. biflexa. This system targets and degrades foreign DNA and we hypothesize that it may contribute to the lower transformation frequency observed in the pathogen relative to the saprophyte. Specifically, we have demonstrated that the CRISPR/cas operon is transcribed during in vitro growth and have integrated part of the operon into L. biflexa and have shown that the genes are also transcribed in this heterologous host. Currently, we are attempting to inactivate specific cas genes in L. interrogans and move the entire operon into L. biflexa. We proceeded in FY2014 to develop a proteomic map of in vitro cultivated L. biflexa to identify highly expressed proteins from membrane- and soluble-fractions. We have identified abundantly-expressed proteins that can be used as cellular markers, as controls for gene expression studies, and also quantified the transcript data from a subset of these genes. Further, we demonstrated that a significant number of L. biflexa proteins are subject to post-translational modification including phosphorylation and acetylation. Highly expressed proteins allow us to identify targets that may play important physiological roles and also use as tagged proteins for various expression studies. This work is being completed with an internal collaboration with Dr. James Carroll in the Laboratory of Persistent Viral Diseases, NIAID and Dr. Lisa Olano of the Research Technologies Branch, NIAID. The long-term objective of this project is to use the improved tools and techniques to understand the basic physiology of leptospires and the mechanisms of infection and pathogenecity of L. interrogans. Together this knowledge should help accelerate the development of preventative measures against Leptospirosis.